Rapid and scalable detection of synthetic mRNA byproducts using polynucleotide phosphorylase and polythymidine oligonucleotides
Production and storage of synthetic mRNA can introduce a variety of byproducts which reduce the overall integrity and functionality of mRNA vaccines and therapeutics. mRNA integrity is therefore designated as a critical quality attribute which must be evaluated with state-of-the-art analytical metho...
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| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Taylor & Francis Group
2024-12-01
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| Series: | RNA Biology |
| Subjects: | |
| Online Access: | https://www.tandfonline.com/doi/10.1080/15476286.2024.2363029 |
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| Summary: | Production and storage of synthetic mRNA can introduce a variety of byproducts which reduce the overall integrity and functionality of mRNA vaccines and therapeutics. mRNA integrity is therefore designated as a critical quality attribute which must be evaluated with state-of-the-art analytical methods before clinical use. The current study first demonstrates the effect of heat degradation on transcript translatability and then describes a novel enzymatic approach to assess the integrity of conventional mRNA and long self-amplifying mRNA. By first hybridizing oligo-T to the poly(A) tail of intact mRNA and subsequently digesting the unhybridized RNA fragments with a 3’-5’ exoribonuclease, individual nucleotides can be selectively released from RNA fragments. The adenosine-based fraction of these nucleotides can then be converted into ATP and detected by luminescence as a sensitive indicator of mRNA byproducts. We developed a polynucleotide phosphorylase (PNPase)-based assay that offers fast and sensitive evaluation of mRNA integrity, regardless of its length, thus presenting a novel and fully scalable alternative to chromatographic-, electrophoresis-, or sequencing-based techniques. |
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| ISSN: | 1547-6286 1555-8584 |